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Reproductive Responses of Common Carp (Cyprinus carpio) Exposed in Cages to Influent of the Las Vegas Wash in Lake Mead, Nevada, from Late Winter to Early Spring E R I N M . S N Y D E R , * ,† S H A N E A . S N Y D E R , †,‡ K E V I N L . K E L L Y , § TIMOTHY S. GROSS,O D A N I E L L . V I L L E N E U V E , †,| SCOTT D. FITZGERALD,⊥ S E R G I O A . V I L L A L O B O S , †,# A N D JOHN P. GIESY† Department of Zoology, National Food Safety and Toxicology Center, and Center for Integrative Toxicology, Michigan State University, 218C Food Safety and Toxicology Building, East Lansing, Michigan 48824, U.S. Bureau of Reclamation, Ecological Research and Investigations (D-8220), Denver Federal Center, P.O. Box 25007, Denver, Colorado 80225, U.S. Geological SurveysBiological Resources Division, Florida Caribbean Science Center, 7920 NW 71st Street, Gainesville, Florida 32653, and Department of Pathobiology and Diagnostic Investigation and Diagnostic Center for Population and Animal Health, P.O. Box 30076, Lansing, Michigan 48909-7576
in LW influent. Male carp caged at LW had slightly greater concentrations of plasma VTG than those at other sites, and a modest elevation in plasma E2 was observed in male carp at LX and LW, but causes other than contaminant exposure cannot be ruled out. Water temperature differences among sites complicated interpretation of the results. Variation in some end points among carp at two different reference sites supports the use of multiple reference sites in field studies of the effects of endocrinedisrupting chemicals.
Introduction Lake Mead is a large reservoir formed by impoundment of the Colorado River behind the Hoover Dam. The reservoir serves as a source of domestic and agricultural water for more than 22 million users (1) and is a popular recreation area. The Las Vegas Wash (LW) delivers tertiary-treated municipal wastewater effluent (87-88%), nonpotable shallow groundwater (6%), and urban runoff (6%) from the urbanized Las Vegas Valley (1, 2) to the Boulder Basin of Lake Mead via the Las Vegas Bay (LX) (Figure 1). LW serves as the sole drainage of the Las Vegas Valley hydrographic basin and contributes approximately 1.5% of the flow into Lake Mead, while the Colorado River provides approximately 97% of the flow, and the Virgin and Muddy Rivers contribute an additional 1.5%.
The Las Vegas Wash (LW) delivers tertiary-treated municipal wastewater effluent, nonpotable shallow groundwater seepage, and runoff from the urbanized Las Vegas Valley to Las Vegas Bay (LX) of Lake Mead. To investigate the potential for contaminants in LW influent to produce effects indicative of endocrine disruption in vivo, adult male and female common carp (Cyprinus carpio) were exposed in cages for 42-48 d at four sites in Lake Mead: LW, LX, and two reference locations in the lake. End points examined included gonadosomatic index; gonad histology; concentrations of plasma vitellogenin (VTG) and plasma sex steroids (17β-estradiol (E2), testosterone (T), 11ketotestosterone (11-KT)); plasma estrogen:androgen ratios (E2:T, E2:11-KT), in vitro production of T by gonad tissue, and hepatopancreas ethoxyresorufin O-deethylase activity. Few differences among fish caged at different sites were potentially attributable to exposure to contaminants
In 1996, the U. S. Geological Survey (USGS) reported that endocrine disruption was occurring in feral adult male and female common carp (Cyprinus carpio) collected in the vicinity of LW and LX (3). Evidence included alterations in plasma sex steroid concentrations in male and female carp relative to those in carp at a reference site and increased concentrations of the estrogen-inducible plasma protein vitellogenin (VTG) in the blood of male carp in the absence of a concomitant increase in plasma 17β-estradiol (E2). The latter indicates that the fish were exposed to an unidentified exogenous estrogenic chemical (4). Previous research has revealed increased concentrations of VTG in the blood of carp (4-10) and other cyprinids (11, 12) exposed to municipal wastewater treatment plant (WWTP) effluent in enclosures or captured from riverine sites influenced by WWTP effluent. Estrogenic chemicals found in municipal WWTP effluent and implicated as potential causative agents for VTG induction in male fish exposed to these effluents include the animal steroids E2 and estrone, the oral contraceptive medication component ethinylestradiol (EE2), and the alkylphenol polyethoxylates (APE) and their degradation products, the alkylphenols (AP) (5, 13-15).
* Corresponding author present address: Black & Veatch Corporation, 4040 S. Eastern Ave., Suite 330, Las Vegas, NV 89119; phone: (702)732-0448; fax: (702)732-7578; e-mail: SnyderEM@ bv.com. † Michigan State University. ‡ Present address: Southern Nevada Water Authority, 1350 Richard Bunker Ave., Henderson, NV 89015. § U.S. Bureau of Reclamation. O U.S. Geological Survey. | Present address: U.S. EPA Mid-Continent Ecology Division, 6201 Congdon Blvd., Duluth, MN 55804-2595. ⊥ Department of Pathobiology and Diagnostic Investigation and Diagnostic Center for Population and Animal Health, Michigan State University, East Lansing, MI 48824. # Present address: Syngenta Crop Protection, Inc., Ecological Toxicology & Environmental Safety, P.O. Box 18300, Greensboro, NC 27419.
Extracts from water samples collected from LW and LX in April 1997 were subjected to a bioassay-directed fractionation and analysis scheme designed to identify the bioactive estrogenic constituents of the LW influent (16). Results indicated that the relatively polar compounds E2 and EE2 were largely responsible for the observed in vitro bioactivity, while relatively nonpolar compounds (polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, organochlorine pesticides) and moderately polar compounds (octylphenol, nonylphenol) were not likely to have contributed significantly to the estrogenic activity (16). The current study was conducted in mid-February through late March to early April of 1999 to determine whether exposure to LW influent, and particularly to estrogenic contaminants found therein, causes reproductive endocrine disruption in adult carp.
10.1021/es049690n CCC: $27.50 Published on Web 11/02/2004
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FIGURE 1. Map of fish cage sites in the Boulder Basin of Lake Mead, NV.
TABLE 1. Cage Deployment and Retrieval Dates and Mean Weights of Carp at Initiation and Completion of the In Situ Exposure Perioda site
sex
HF HF LW LW LX LX MC MC WB WB
male female male female male female male female male female
final mean date date initial mean deployed retrieved wt per fish (g)b wt per fish (g)c na na 02/17 02/17 02/18 02/18 02/16 02/16 02/15 02/15
na na 04/05 04/05 04/07 04/07 04/01 04/01 03/29 03/29
na na 163 123 187 153 184 141 173 136
160 107 159 114 178 115 189 123 171 124
sites. Two cages were placed directly at the point (site LW) where LW enters LX such that the carp received maximum exposure to the influent. Two cages were placed further out in LX at a point (site LX) where the LW influent was more dilute but still readily detected by its greater conductivity and turbidity than main body lake water. Cages (each 2.1 m × 2.1 m × 1.4 m deep) were suspended approximately 1.41.7 m below the surface of the water to prevent sunburn and to avoid boat traffic and vandalism. Fish were fed approximately twice weekly by dropping a sinking pellet food (Silver Cup trout pellets; Nelson & Sons, Inc.; Murray, UT) through the top of each cage. Fish were observed feeding on pellets at the reference sites, but greater turbidity prevented observation of fish at LW and LX.
Materials and Methods
Water Quality. Water quality was monitored periodically at each exposure site. Water temperature, pH, dissolved oxygen (DO), conductivity, and turbidity were measured using a Hydrolab Surveyor 4 data recorder equipped with a H2O Sonde water quality multiprobe (Hydrolab Corporation, Austin, TX). Measurements taken at 1, 2, and 3 m depths at each site were averaged on each sampling day to cover the entire span of the fish cages. Water samples for measurement of alkalinity, hardness, total ammonia, and nitrite were collected with a horizontal water sampler (Wildlife Supply Company, Saginaw, MI) at the approximate mid-depth of the cages at each site, and the water quality parameters were measured with Hach test kits (Hach Company, Loveland, CO).
Caged Fish. Sexually mature, adult male and female common carp (2-3 yr of age) were purchased from J&J Aquafarms (Sanger, CA). Males and females were held in separate cement holding ponds at the Lake Mead Fish Hatchery, Boulder City, NV, for 4-7 d prior to placing them into cages in Lake Mead. While fish were held at the hatchery, they were fed daily ad libitum with a floating dense culture pellet food for pond fish (Aquatic Ecosystems, Apopka, FL). Some carp were retained at the hatchery as hatchery references (HF) to determine initial concentrations of plasma VTG and stage of sexual maturation of the fish at the beginning of the study. In situ exposures began in mid-February and lasted through late March or early April (Table 1). Two fish cages (Aquatic Ecosystems, Apopka, FL), one containing 30 male carp and one containing 30 female carp, were deployed at each of four sites in Lake Mead (Figure 1). Two sites, Water Barge Cove (WB) and Moon Cove (MC), are not influenced significantly by the flow of LW and were considered reference
Fish Retrieval and Sample Collection. The exposure durations for pairs of cages at different sites were: LW, 47 d; LX, 48 d; WB, 42 d; and MC, 44 d. At the end of the exposure period, the carp were transported in aerated livewells a short distance to shore, where they were dispatched by immersion in 200 mg/L tricaine methanesulfonate (Tricaine-S or MS222; Aquatic Ecosystems, Apopka, FL). Blood was collected immediately from the caudal vasculature of each fish using a chilled, heparinized needle and syringe. For each carp, up to 3 mL of blood was placed into a chilled polyethylene centrifuge tube pretreated with a protease inhibitor (aprotinin; Sigma, St. Louis, MO) for a final concentration of 1 TIU (trypsin inhibitor unit) per mL of blood. To minimize variation in plasma sex steroid concentrations caused by diel cycling, blood samples were collected from fish of the same sex at different sites within the same 2-3-h time period (17). Gentle pressure was applied to the abdomen of male fish to determine whether milt was running. Each fish was weighed
a
wt, weight; na, not applicable; LW, Las Vegas Wash; LX, Las Vegas Bay; MC, Moon Cove; WB, Water Barge Cove; HF, Hatchery Fish (hatchery controls). b Calculated by dividing initial total weight of fish (in kg) by the number of fish weighed (30 per cage) and converting to mean weight in grams per fish. Initial total weight of carp placed in each cage was determined to the nearest 0.01 kg by weighing a bucket of water on a scale, then adding the fish to the bucket and re-weighing. c Calculated by dividing final total weight of fish (in kg) by number of fish present when the cage was retrieved and converting to mean weight in grams per fish. Final total weight was determined by summing the individual fish weights recorded after cage retrieval.
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to the nearest 1 g and measured for standard length to the nearest 0.1 cm. Fish carcasses and blood samples were transported on ice to the laboratory. The sex of each fish was confirmed by gross observation of the gonads. A random subsample of 16 fish from each cage was selected for measurement of gonad weights, collection of hepatopancreas samples and tissues for histological examination, and in vitro gonad incubations. Gonads were removed and weighed to the nearest 0.001 g with a portable analytical balance. The caudal half of one gonad (right side) from each fish was preserved in 10% neutral-buffered formalin. Samples of hepatopancreas tissue were frozen immediately in liquid nitrogen for subsequent measurement of ethoxyresorufin O-deethylase (EROD) activity. In vitro incubations of gonad tissue for measurement of basal testosterone (T) production (in vitro T production) were conducted using methods similar to those described previously (18). For each female carp, 12 × 75 mm test tubes, each containing 20 vitellogenic follicles (0.7-1.1 mm diameter) in 0.5 mL of medium 199 without phenol red (Sigma, St. Louis, MO), were incubated in triplicate at 16-17 °C for 18 h. For male carp, a preweighed portion of testicular tissue (18-25 mg) was incubated in the same manner as the ovarian follicles. Blood samples were centrifuged at 3000g for 10 min at 4 °C. Plasma was collected, divided into several aliquots per sample, and frozen at -80 °C. Reference carp maintained at the fish hatchery (HF) were sampled in the same manner 14 d after the last fish cages were deployed in the lake. Gonad Histology. From each preserved gonad sample, a section that represented the centermost portion of the organ was embedded in paraffin, sectioned at 5 µm, and stained with haematoxylin and eosin. Gonad samples from 12 to 16 male carp per site and from 12 to 16 female carp per site were examined histologically, as described previously (19, 20), and testes were designated as spermatogenically active or inactive. Plasma VTG. Plasma samples were analyzed for VTG with a competitive enzyme-linked immunosorbent assay (ELISA) described elsewhere (21). Inter-assay percent coefficient of variation was 14.2% for a single blood plasma sample analyzed in each ELISA run. Sex Steroids and EROD Activity. Concentrations of the sex steroid hormones E2, T, and 11-ketotestosterone (11KT) were measured in blood plasma and concentrations of T were measured in gonad incubation medium by competitive radioimmunoassay as described elsewhere (22). EROD activity in postmitochondrial supernatant prepared from hepatopancreas tissue samples was measured using a method adapted from previous methods (21, 23, 24). Calculation of Indices and Statistical Analyses. Gonadosomatic index (GSI) was calculated for each fish as GSI ) [gonad weight/(body weight - gonad weight)], where all weights are in grams. Fulton-type condition factor (K) was calculated for each fish as K ) (W/L3) × 10 000, where W) weight in g and L ) standard length in mm. Data were analyzed with the aid of SYSTAT Version 9 for Windows (SPSS Science, Chicago, IL; 1998), the SAS System for Windows Release 7.00 (SAS Institute, Cary, NC; 1998), and SAS JMP Version 4.0.4 (SAS Institute, Cary, NC; 2001). When data met the assumptions of parametric statistical methods, differences among sites and between sexes were examined by one-way or two-way analysis of variance (ANOVA) followed by Tukey’s HSD post-hoc comparison of means. When log transformation failed to cause data to meet the assumptions of parametric methods, a Kruskal-Wallis test was approximated by examining the ranks of the data by ANOVA, followed by a Tukey-like test conducted on the ranks of the data. A Student’s t-test or Mann-Whitney test was used to explore differences in some end points between male and female carp held in cages at the same site. Effects were considered
to be statistically significant at the 0.05 level of type I error (R) for all analyses. In the few cases when plasma samples contained VTG concentrations less than the method detection limit (MDL) of 0.267 µg of VTG/mL, the value of one-half the MDL was substituted for statistical analyses. As in other fish species, gonadal sex steroid synthesis, concentrations of plasma sex steroids, and EROD activity vary with the stage of sexual maturation in carp (22, 25, 26). Therefore, comparisons of plasma sex steroid concentrations, in vitro T production, plasma VTG (because it is dependent on plasma E2, at least in females), and EROD activity among fish held in cages at different sites were restricted to those carp determined to be in a similar stage of sexual maturation within each sex. Spermiating males with running milt and testes determined by histological examination to be spermatogenically active were considered to be in the same stage of sexual maturation. Females with ovaries containing more than 50% primary follicles and containing some intact tertiary follicles were considered to be in the same stage of sexual maturation. Multivariate profile analysis (27) conducted with SAS was used to examine patterns of ovarian follicles in different stages of development (primary, secondary, or tertiary). The analysis was conducted on arcsine-transformed proportions of follicles (parametric) and also on the ranks of these proportions (nonparametric). The arcsine transformation was executed to cause proportions to assume a nearly normal distribution (28). Because this transformation does not perform well at the extreme ends of the range of possible values, for the proportions X/n (where X ) the number of follicles in a particular condition and n ) 50 follicles counted), the arcsine transformation was improved by replacing 0/n with 1/4n and n/n with 1-1/4n, as suggested by Zar (28).
Results and Discussion One of the major challenges in determining whether animals are experiencing endocrine disruption in the environment is deciding whether statistically significant changes constitute biologically significant changes, with negative consequences at the organism or population levels. It is particularly difficult to assess whether changes in plasma sex steroid or VTG levels can be considered to be adverse impacts because normal ranges have not been established for most fish species, particularly for wild fish. Factors such as temperature, photoperiod, nutritional status, age, season, reproductive cycles, and diel cycles can affect reproductive end points, including circulating sex steroid concentrations. In this study, the effects of these factors were minimized, and they were considered in the interpretation of the results. Results of this study are compared to previously reported baseline information on reproductive end points in common carp. However, data from different studies were not collected under a single standardized protocol, so caution should be used in making comparisons. Variation in reproductive end points can occur among cyprinid fish at different sites regardless of potential impacts due to pollution (22, 29, 30); therefore, two reference sites were included in this study. Water Quality. Water quality parameters measured at each site were generally within tolerance guidelines for optimal health and growth of cyprinid fish (Table 2) (31). Water hardness at all sites reached levels greater than the optimum range for carp, but they can tolerate hardness exceeding 250 mg/L (31). Hardness was greater at sites LX and LW than at the reference sites. There were consistent water temperature differences among sites (LW > LX > WB > MC), with average site temperatures ranging from 17.1 °C at LW to 13.3 °C at MC. Conductivity measurements were greatest at LW, less at LX, and least at WB and MC, indicating that carp caged at LW were receiving the greatest exposure and those caged at LX were receiving less exposure to Las VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Water Quality Parameters Measured at Exposure Sites for Carp Held in Cages in Lake Mead, NVa water quality parameter temperatureb (°C) pHb DOb (mg/L) conductivityb (µS) turbidityb (NTU) alkalinityb (mg/L) hardnessc,d (mg/L) tot ammoniac (mg/L)
site LW
LX
MC
WB
17.1 (16.2-18.7) 8.38 (8.10-8.53) 12.02 (9.58-13.31) 1561 (1423-1636) 3.4 (2.8-4.1) 108 (107-108) 348 (336-360) 0.1 (0.0-0.2)
16.0 (15.3-16.9) 8.40 (8.08-8.61) 11.66 (9.57-12.83) 1250 (1088-1464) 2.0 (0.8-3.7) 107 (105-108) 333 (306-360) 0.2 (0.0-0.3)
13.3 (12.6-14.2) 8.14 (8.05-8.23) 9.79 (9.58-10.13) 903 (893-922) 1.6 (0.0-4.6) 109 (107-110) 270 (268-272) 0.2 (0.0-0.3)
14.5 (14.0-14.9) 8.06 (8.00-8.11) 9.70 (9.57-9.83) 889 (883-895) 0.4 (0.4-0.4) 110 (110-110) 202 (134-270) 0.1 (0.0-0.2)
a All measurements were made between March 5 and March 26, and sampling days were spread throughout the carp exposure period. Reported values are means, with ranges in parentheses. DO, dissolved oxygen; tot ammonia, total ammonia; LW, Las Vegas Wash; LX, Las Vegas Bay; MC, Moon Cove; WB, Water Barge Cove. b Measurements were taken at 1, 2, and 3 m depths at each site to span the depths of the cages, and a mean was calculated for each sampling day. Reported values are grand means for three sampling days per site. c Reported values are means for two measurements conducted on different sampling days. d As CaCO3 equivalents.
Vegas Wash influent. Conductivity measurements at the reference sites were consistent with that of main body lake water. Nitrite (MDL ) 2 mg/L) was not detected in any of the water samples. Survival and Condition. Four carp died during the exposure: two males at WB, one male at LX, and one female at LX. The number of fish that died was small in comparison with other studies in which fish were held in municipal WWTP effluents (5, 32, 33) or in rivers receiving municipal WWTP effluents (15, 29, 34). With the exception of male carp held at MC, the mean weight per fish in each cage was less at the end of the exposure than it was at the beginning (Table 1), possibly indicating that the fish were stressed by the exposure conditions. However, this might be an artifact of the different methods used to collect initial and final mean weight data (see legend for Table 1). Mean K for female carp at LX was less than that for females at both reference sites (Table 3). The reason for this difference is not clear, but the difference is small, and the fish at LX did not appear to be unhealthy. Male carp at LW and LX had K values similar to those for males from HF and MC (Table 3). Results were not different when statistical analysis of K values was restricted to carp in the same stage of sexual maturation. Gonad Development and Histology. Previous studies have demonstrated decreased GSI in male, female, and intersex fish exposed to municipal WWTP effluent (12, 14). After repeated seasonal sampling of feral common carp from Las Vegas Bay and from the Overton Arm of Lake Mead, a location not influenced by the influent of LW and thus considered a reference site, Patin ˜ o et al. found that males from Las Vegas Bay consistently had smaller GSI (35). In the current study, no statistically significant differences in mean GSI occurred among female carp held in cages at the lake sites, but mean GSI of female carp at HF was significantly less than that of females at each of the lake sites (Table 3). No statistically significant differences in mean GSI were observed among male carp held at different sites, including HF (Table 3). Results were similar when statistical analyses were restricted to females or males in the same stage of sexual maturation. Adult fathead minnows exposed to waterborne E2 showed changes in the proportions of ovarian follicles in different stages of development, with greater exposure concentrations resulting in the appearance of atretic follicles and the absence 6388
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of mature (tertiary) follicles (20). In the current study, there were no statistically significant differences in ovarian follicle development profile among carp held at different lake sites (Figure 2), indicating that they were in the same stage of ovarian development. Ovaries of all caged carp contained a preponderance of primary follicles and more tertiary than secondary follicles. Incidence and severity of oocyte atresia were low. Only five of the 63 caged carp subsampled for histological examination demonstrated atretic ovarian follicles, and in each case, only one follicle per 50 examined in one fish was atretic. Carp with atretic ovarian follicles were evenly distributed among the exposure sites. Fish undergoing active gonadal growth and development, particularly those in the middle stages of gonadal recrudescence, provide the most useful information regarding the end points examined in this study. Sex steroid synthesis and gonadal growth occur during that time, making it possible to detect decreases in circulating sex steroid concentrations or in GSI caused by exposure to endocrine-disrupting chemicals (EDCs), while variability and sensitivity to siterelated differences in reproductive timing unrelated to contaminant exposure are minimized (17). The carp held at HF were sampled earlier than the carp held at the lake sites and initially were assumed to be representative of the condition of carp held in cages prior to their deployment in the lake with respect to GSI and plasma VTG concentrations. The lesser GSI in female carp held at HF relative to those in females held in the lake suggests that the latter were experiencing gonadal growth and development during the exposure. However, proportions of primary follicles were less and proportions of secondary follicles were greater in ovaries of female carp held at HF. This difference was not anticipated and warrants caution in making comparisons between HF females and those caged in the lake. Mean GSI for female carp held in cages at all sites in the lake (Table 3) indicate that the majority of female carp were in the middle to late stages of gonadal recrudescence and probably not ripe for spawning, when GSI could be expected to be approximately 17-28% (36-40), although lesser GSI have been reported for ripe female carp (41, 42). The average water temperatures measured at the exposure sites were less than the optimal range of 19-24 °C that typically induces carp to spawn in temperate regions (42-44). No indications of ovulation (36, 37) were apparent during gross examination,
TABLE 3. Condition Factor (K), Gonadosomatic Index (GSI), In Vitro Testosterone (T) Production, and Hepatopancreas Ethoxyresorufin O-Deethylase (EROD) Activity in Adult Female and Male Carp Held in Cages in Lake Meada females
males mean
Tukey group
0.21-0.28 0.22-0.33 0.22-0.28 0.23-0.36 0.23-0.29
0.25 0.24 0.24 0.26 0.26
Condition Factor (K) abc HF ab LW a LX c MC bc WB
12 29 29 28 28
12 16 16 15 15
0.022-0.150 0.065-0.207 0.081-0.190 0.060-0.218 0.030-0.179
0.085 0.134 0.141 0.154 0.130
Gonadosomatic Index (GSI) a HF b LW b LX b MC b WB
LW LX MC WB
15 16 13 14
65-119 137-387 81-194 84-160
82 239 121 120
In Vitro T Production a LW c LX b MC b WB
HF LW LX MC WB
10 15 16 13 14
5.3-15 4.2-37 4.7-25 2.6-13 4.1-14
Hepatopancreas EROD Activity (pmol mg-1 min-1) 9.5 a HF 12 18 b LW 15 11 a LX 16 7.8 a MC 16 8.8 a WB 15
site
n
HF LW LX MC WB
12 30 27 28 29
HF LW LX MC WB
range
site
mean
Tukey group
0.22-0.25 0.19-0.28 0.19-0.26 0.18-0.27 0.23-0.30
0.24 0.23 0.23 0.24 0.26
a a a ab b
12 16 16 16 15
0.025-0.062 0.016-0.069 0.018-0.096 0.025-0.099 0.012-0.086
0.046 0.050 0.054 0.056 0.062
a a a a a
15 16 16 15
3.8-5.9 6.8-16 4.0-9.7 3.8-8.8
5.0 12 5.8 5.9
a b a a
8.5-22 7.7-50 10-29 1.2-25 1.5-27
12 24 16 8.8 12
abc c bc a ab
n
range
a Fulton-type condition factors (K) are reported for all caged carp, regardless of stage of sexual maturation. K values were calculated as K ) (W/L3) × 10 000, where W ) weight in g and L ) standard length in mm. Statistical analyses were conducted on log-transformed K values for both sexes. GSI are reported for all fish for which gonad weight was measured. GSI ) [gonad weight/(body weight - gonad weight)], where all weights are in g. Statistical analyses were conducted on untransformed GSI values for both sexes. In vitro T production is reported only for those carp in the same stage of sexual maturation within each sex. For female carp, in vitro T production per 20 ovarian follicles (pg of T/20 follicles) is reported. For male carp, in vitro T production per mg of testicular tissue (pg of T/mg of tissue) is reported. Statistical analyses were conducted on log transformed in vitro T production values for both sexes. Statistical analyses were conducted on log transformed hepatopancreas EROD activity values (pmol mg-1 min-1) for both sexes. Within each combination of end point and sex, fish at sites marked with common letters (Tukey groups) were not significantly different. Differences were considered to be significant at p < 0.05. LW, Las Vegas Wash; LX, Las Vegas Bay; MC, Moon Cove; WB, Water Barge Cove; HF, Hatchery Fish (hatchery controls).
stages of gonadal recrudescence at the end of the exposure period.
FIGURE 2. Profiles of percentages of follicles in each of three stages (primary, secondary, and tertiary) in the ovaries of adult female carp held in cages in Lake Mead. Each stacked bar represents the mean percentages of follicles in the corresponding stages. Numbers above bars are sample sizes. LW, Las Vegas Wash; LX, Las Vegas Bay; MC, Moon Cove; WB, Water Barge Cove. and no post-ovulatory follicles were observed during histological examination of the ovaries. Although the difference was not statistically significant, the lesser mean GSI for male carp held at HF suggests that the male carp caged in the lake were undergoing active gonadal growth during the exposure period. On the basis of GSI reported in male common carp or mirror carp in different stages of sexual maturation (36-40, 45, 46), most of the male carp used in this study appeared to be in the middle to late
All of the male carp used in this study had spermatogenically active testes, and no degenerative changes were observed during histological examination of the testes. Only two male carp did not produce running milt (one at MC and one at LW) at the end of the exposure period. Sertoli cell proliferation was observed previously in testes of fathead minnows exposed to the natural estrogen E2 or to the estrogenic chemical 4-nonylphenol (NP) (19, 20). Median Sertoli cell proliferation scores were not significantly different among male carp held in cages at different sites in the lake or at HF. Only two male carp, one caged at LX and one at LW, exhibited Sertoli cell proliferation. Both of these males had only mild lesions, but the one caged at LW failed to produce running milt. Although it is possible that these lesions were related to contaminants in LW influent, the low incidence and mild presentation provide little support for that conclusion. Four male carp were incorrectly presumed to be females at the beginning of the study due to their failure to release milt and lack of any other indication of male gender (nuptial tubercles, male coloration) and were placed in cages designated for females. None of these fish produced milt at the end of the exposure period, and three (one at each of the following sites: LX, MC, and WB) were later examined histologically and found to contain spermatogenically inactive testes. The fourth fish, placed with the females at LX, was not examined histologically. Because some of the carp used in this study were only 2 years old, it is possible that a few had not reached sexual maturity prior to being placed VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Concentrations of plasma vitellogenin (VTG, µg/mL) in adult female carp held in cages in Lake Mead. Data are reported only for carp in the same stage of sexual maturation. Closed circles represent individual data points, and bar heights represent site medians. Sample sizes are as follows: HF ) 10, LW ) 15, LX ) 16, MC )13, WB ) 14. Nonparametric statistical analyses were conducted, and letters above bars represent Tukey-like groupings (p < 0.05); i.e., fish at sites with bars marked with common letters were not significantly different with regard to plasma VTG concentration. LW, Las Vegas Wash; LX, Las Vegas Bay; MC, Moon Cove; WB, Water Barge Cove; HF, Hatchery Fish (hatchery controls). in the cages. All of the other male carp examined were actively spermatogenic, and most were spermiating (producing running milt). It is unlikely that the presence of the undeveloped males affected the reproductive condition of the females with which they were held. Intersex Carp. One carp placed with the females at reference site MC was determined to be intersex upon gross and histological examination, with approximately half testicular tissue and half ovarian tissue in one gonad. It is unlikely that this single occurrence of intersex gonad resulted from exposure to chemicals in the environment during the study period. The fish were sexually differentiated adults prior to commencement of the study, and concentrations of EDCs sufficient to produce intersex gonads in adult fish were not likely to have been encountered at the reference site. Occasionally, intersexuality appears to occur spontaneously under natural conditions in cyprinid fish (47). Plasma VTG in Females. In male fish or in sexually immature female fish, elevated plasma VTG concentration is an indicator of exposure to estrogenic chemicals. Reduced plasma VTG, possibly indicating endocrine disruption, has been reported in sexually mature female carp collected downstream from municipal WWTP effluent discharges (26). Concentrations of VTG in blood plasma of all female carp used in this study ranged from 0.024 to 29.7 mg of VTG/mL. There were no statistically significant differences in plasma VTG concentration among female carp held in cages at different sites in Lake Mead (Figure 3), but only females at LX had elevated plasma VTG concentrations relative to those in females held at HF, which might be representative of initial plasma VTG concentrations in the caged carp. Conclusions regarding site-to-site differences were the same regardless of whether the analysis was restricted to carp in the same stage of sexual maturation. In a study of feral Lake Mead carp, females caught in LX had greater plasma VTG concentrations than those caught in LW or at a reference site (3). If greater plasma VTG concentrations in female carp at LX are due to waterborne contaminants, it is not clear why the more dilute influent of the Las Vegas Wash at LX might produce greater plasma VTG concentrations than the more concentrated influent at LW. Excluding reference site WB, plasma E2 (Figure 4) and plasma VTG concentrations in female carp varied together among the Lake Mead sites, although differences among females held at different sites in the lake were not statistically 6390
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FIGURE 4. Concentrations of plasma sex steroids 17β-estradiol (E2), testosterone (T), and 11-ketotestosterone (11-KT) in adult female carp held in cages in Lake Mead. All concentrations are in pg/mL. Data are reported only for those carp in the same stage of sexual maturation. Bar heights represent site means, and error bars are 2 SEM. Sample sizes are as follows: LW ) 15, LX ) 16, MC ) 13, WB ) 14. Nonparametric statistical analyses were conducted on E2 and 11-KT data. Parametric statistical analyses were conducted on log transformed T data. Letters of the same style above bars represent Tukey or Tukey-like groupings (p < 0.05) for a single steroid; i.e., fish at sites with bars marked with common letters of the same style were not significantly different with regard to the steroid of interest. LW, Las Vegas Wash; LX, Las Vegas Bay; MC, Moon Cove; WB, Water Barge Cove. significant for either E2 or VTG. Concentrations of plasma VTG in female carp were generally within the range of concentrations previously reported for female carp collected in a national reconnaissance study in the United States (22) or collected from reference sites in rivers in Spain (8) and were similar to those previously reported for female carp collected in the spring from a reference site in Lake Mead (less than 10 mg/mL) (3). Overall, these findings indicate that plasma VTG concentrations in female carp held in cages in Lake Mead were not significantly affected by LW influent. Plasma VTG in Males. Plasma VTG concentrations in male carp ranged from less than the MDL (0.267 µg of VTG/mL) to 51.8 µg of VTG/mL for all male carp used in this study and for males in the same stage of sexual maturation. The median plasma VTG concentration in male carp at LW was significantly greater (3-10-fold) than that of males at the other sites. The number of nondetects was small at each site: HF (1/12), MC (1/28), WB (2/28), LX (1/29), and LW (0/29). When the analysis was restricted to carp in the same stage of sexual maturation, conclusions regarding site-to-site differences were the same, and the number of nondetects per site remained small: HF (1/12), MC (1/15), WB (1/16), LX (1/16), and LW (0/15). Greater plasma VTG concentrations in male carp at LW were accompanied by greater plasma E2 concentrations (Figure 5 and 6) relative to those in males at both reference sites. However, male carp held at LX or at HF had similar or greater mean concentrations of plasma E2 but did not exhibit an increase in plasma VTG. This finding implies that male carp held at LW were exposed to an exogenous estrogenic substance that produced an increase in plasma VTG independent of endogenous E2. Alternatively, plasma E2 in the fish at LX and HF might have been bound to a greater extent by plasma binding proteins and less available to hepatocytes (48). Water temperature also must be considered as a factor potentially influencing VTG induction. In a study by Hernandez et al., plasma VTG was induced in male carp given i.p. injections of E2 when they were held at 18-20 °C, but there was no detectable response at 9-10 °C (49). The water temperature at LW was slightly greater than the temperature at LX (average 17.1 and 16.0 °C, respectively). It is not known
FIGURE 5. Concentrations of plasma vitellogenin (VTG, µg/mL) in adult male carp held in cages in Lake Mead. Data are reported only for carp in the same stage of sexual maturation. Closed circles represent individual data points, and bar heights represent site medians. Sample sizes are as follows: HF ) 12, LW ) 15, LX ) 16, MC ) 16, WB ) 15. Nonparametric statistical analyses were conducted, and letters above bars represent Tukey-like groupings (p < 0.05); i.e., fish at sites with bars marked with common letters were not significantly different with regard to plasma VTG concentration. LW, Las Vegas Wash; LX, Las Vegas Bay; MC, Moon Cove; WB, Water Barge Cove; HF, Hatchery Fish (hatchery controls). whether the slight differences in temperature were sufficient to explain differences in plasma VTG among male carp at different sites. The elevated plasma VTG concentrations in male carp held at LW are not as great as those reported for feral male carp caught at LX and LW in 1995 (3). Low milligrams per milliliter concentrations of plasma VTG were reported in male carp exposed to the influent of LW, as opposed to concentrations less than the MDL of 1 mg of VTG/mL in male carp caught at a reference site (3). Elevated concentrations and relative induction of plasma VTG in male carp held at LW were not dramatic when compared to results from some other studies of male carp exposed to municipal WWTP effluent or to other potential sources of contamination elsewhere in the United States (4) and in Japan (10). However, in research conducted in Spain with wild adult carp collected from rivers, more modest increases in plasma VTG concentration were associated with exposure to municipal WWTP effluents (7-9). Plasma VTG concentrations in male carp in the current study fall within the range of concentrations previously reported for male carp collected upstream of WWTP discharges (25), for apparently normal male carp collected from rivers (6), and for male carp sampled as laboratory controls (40, 50-52). In a recent study in Spain, plasma VTG reached approximately 4-10 µg of VTG/mL in adult male carp collected from reference sites upstream of municipal WWTP discharges in rivers in March and May, when water temperatures spanned those observed in our study (8). Median plasma VTG concentration for female carp was significantly greater than that for males within each site. Concentrations of VTG in the plasma of male and female carp held in cages at the same site overlapped slightly and only within reference site WB. In contrast, concentrations of plasma VTG in feral male carp collected from LX and LW in 1995 overlapped substantially with concentrations in females (3). Likewise, Folmar et al. reported overlapping plasma VTG concentrations in male and female common carp collected from a municipal WWTP effluent canal where the fish also demonstrated alterations in sex steroid concentrations that were indicative of endocrine disruption (4). That VTG in males was induced to levels seen in females might indicate a more severe effect on male carp than was observed in the current study.
FIGURE 6. Concentrations of plasma sex steroids 17β-estradiol (E2), testosterone (T), and 11-ketotestosterone (11-KT) in adult male carp held in cages in Lake Mead. All concentrations are in pg/mL. Data are reported only for those carp in the same stage of sexual maturation. Bar heights represent site means, and error bars are 2 SEM. Samples sizes are as follows: LW ) 15, LX ) 16, MC ) 16, WB ) 15. Parametric statistical analyses were conducted on untransformed E2 data and on log transformed T and 11-KT data. Letters of the same style above boxes represent Tukey groupings (p < 0.05) for a single steroid; i.e., fish at sites with bars marked with common letters of the same style were not significantly different with regard to the steroid of interest. LW, Las Vegas Wash; LX, Las Vegas Bay; MC, Moon Cove; WB, Water Barge Cove. Plasma Sex Steroids in Males. The androgens 11-KT and T are dominant sex steroids in male fish including carp (53), and 11-KT is often considered to be the principal androgen in carp (53-55). E2 also occurs in the blood plasma of male fish. Changes in circulating sex steroid concentrations have been documented in male fish exposed to municipal WWTP effluents, but the effects are not consistent. Male walleye collected from a natural side channel of the Mississippi River receiving municipal WWTP effluent had elevated serum E2 concentrations (56), but E2 concentrations were not affected in male carp collected from the same area (4). In those same two studies, male walleye and carp exposed to municipal WWTP effluent had depressed serum T concentrations. In May 1995, feral male carp collected at LX, but not those collected at LW, had depressed plasma E2 and plasma 11-KT concentrations relative to males from a reference site (3); plasma T concentrations were not measured. Plasma E2 concentrations in male carp ranged from 134 to 649 pg of E2/mL for all male carp used in the current study and for males in the same stage of sexual maturation. Male carp held in cages at LW and LX had plasma E2 concentrations greater than those in males held at the reference sites (Figure 6). Plasma E2 concentrations in male carp held at the reference sites were similar to or slightly greater than those reported previously for male carp outside the spawning season (4, 22, 25, 57-60). Even the greater concentrations of plasma E2 in male carp at LW and LX in the current study are comparable to, and generally less than, concentrations measured in feral male carp captured in May 1995 from a reference site in Lake Mead (3). The elevation in E2 concentrations in males at LW and LX relative to the reference sites is modest as compared to the larger changes in plasma E2 that may take place over the reproductive cycle of male carp. Basal concentrations of plasma E2 less than 0.3 ng/mL, and significant peaks at 1-2 ng/mL during the spawning season have been reported (57). Concentrations of T in plasma of male carp ranged from 213 to 3462 pg of T/mL for all male carp used in the study and from 241 to 3462 pg of T/mL among male carp in the same stage of sexual maturation. There were no statistically significant differences in plasma T concentrations among males held at different sites in the lake (Figure 6), and site VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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means were on the lower end of the range of normal concentrations of plasma T previously reported for male carp outside the spawning season (4, 57-62). Concentrations of 11-KT in plasma of male carp ranged from 277 to 4364 pg of 11-KT/mL for all male carp used in the current study and for male carp in the same stage of sexual maturation. There were no statistically significant differences in plasma 11-KT concentrations among male carp held in cages at LW, LX, and reference site WB (Figure 6), and concentrations were similar to those reported for feral male carp captured from LW and from a reference site in Lake Mead (3). Plasma 11-KT concentrations in male carp in the current study also were greater than or similar to concentrations previously reported for male carp outside the spawning period (25, 58, 62). Relative proportions of plasma E2, T and 11-KT concentrations in male carp were similar among sites (Figure 6), so absolute steroid concentrations might reflect site-to-site differences in steroidogenic capacity related to water temperature. There were no differences in E2:11-KT ratio or E2:T ratio among sites that would indicate an imbalance of the male sex steroid milieu (see below). Plasma Sex Steroids in Females. E2 and T are important sex steroids in female teleosts (18). During gonadal recrudescence, T serves as a precursor for E2, which in turn induces vitellogenesis (18). Although 11-KT has been detected in the plasma or serum of the females of a wide variety of teleost fish species, including koi carp (63) and common carp (3, 22, 25, 58), the functional significance of this hormone in female fish is unknown. Elevated plasma 11-KT concentrations in female common carp have been cited previously as evidence of endocrine disruption (3), and plasma 11-KT concentrations in female carp collected from streams in the United States were reported to be positively associated with exposure to dissolved pesticides (22). Plasma E2 concentrations ranged from 92 to 1289 pg of E2/mL for all females used in this study and for females in the same stage of sexual maturation. Plasma E2 concentrations in female carp held in cages at LW and LX were not significantly different from those in females held at the MC reference site (Figure 4). Plasma T concentrations in female carp ranged from 183 to 2660 pg of T/mL for all female carp used in the study and from 198 to 2660 pg of T/mL for females in the same stage of sexual maturation. Although there were statistically significant differences in plasma T among females held in cages at different sites (WB and LW < LX < MC) (Figure 4), the differences do not appear to be related to LW influent. Plasma E2 and T concentrations in female carp held in cages at all sites fell within the range of concentrations reported elsewhere for female carp captured from reference sites in rivers (25) or held in aquaria under simulated natural photoperiod (58), in outdoor tanks (62), or in ponds (64). Plasma E2 concentrations in female carp also were similar to those reported for feral female carp collected in May 1995 from a reference site in Lake Mead (3). Plasma 11-KT concentrations ranged from 64 to 3632 pg of 11-KT/mL for all female carp used in the study and from 64 to 667 pg of 11-KT/mL among female carp in the same stage of sexual maturation. Female carp held at LW and LX had plasma 11-KT concentrations similar to those in females at both reference sites (Figure 4), and concentrations in most female carp were less than 0.7 ng/mL. Site median plasma 11-KT concentrations for female carp were less than concentrations reported for a captive stock of Japanese koi carp (0.61 ng/mL, SEM ) 0.20, n ) 4) (63) or for feral female carp captured from Lake Mead (3) and were within the range of plasma 11-KT concentrations reported for female carp captured in the fall in the Western United States (22), held in aquaria under simulated natural photoperiod in spring (58), or captured from reference sites in streams (25). 6392
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FIGURE 7. Ratio of plasma 17β-estradiol to plasma 11-ketotestosterone (E2:11-KT) in adult female and male carp. Data are reported only for those carp in the same stage of sexual maturation. Bar heights represent site means, error bars are 2 SEM, and numbers on bars are sample sizes. Log transformed data were subjected to parametric statistical analyses for both sexes. Letters above bars represent Tukey groupings (p < 0.05) within each sex; i.e., fish at sites with bars marked with common letters were not significantly different with regard to E2:11-KT ratio. LW, Las Vegas Wash; LX, Las Vegas Bay; MC, Moon Cove; WB, Water Barge Cove; HF, Hatchery Fish (hatchery controls). Plasma Estrogen to Androgen (E:A) Ratios. Plasma concentrations of androgens are typically greater than concentrations of estrogens in male carp, while the opposite is true for female carp (3, 22), so estrogen to androgen (E:A) ratios typically are less than 1 for males and greater than 1 for females (with the exception of E2:T in females during the spawning periodssee below). E:A ratios (E2:T ratios or E2:11-KT ratios) appear to be sensitive indicators of abnormal sex steroid milieu and to be better indicators of the reproductive health of fish than the concentrations of individual sex steroids (29), since the ratios tend to exhibit differences between male and female fish regardless of absolute steroid concentrations. Plasma E2:11-KT ratios ranged from 0.14 to 1.13 for all male carp used in this study and for males in the same stage of sexual maturation. There were no statistically significant differences in E2:11-KT ratio among males held at different sites (Figure 7). All males in the same stage of sexual maturation had E2:11-KT ratios less than 1, except for one at LW (1.13). Mean E2:11-KT ratio for females exceeded that for males within each site by more than 2-9-fold. Plasma E2:11-KT ratios ranged from 0.17 to 5.16 in all female carp used in the study and from 0.31 to 5.16 for females in the same stage of sexual maturation. Female carp caged at LW and LX did not differ significantly from those at MC with regard to plasma E2:11-KT ratio (Figure 7). E2:11-KT ratios were greater than 1 for most female carp in the same stage of sexual maturation, with the exception of those at site WB, where most females (13/14) had E2:11-KT ratios less than 1.0. Only three other female carp in the same stage of sexual maturation, one at LW (0.87) and two at MC (0.95 and 0.99), had E2:11-KT ratios less than 1.0, indicating a normal state for most female carp in the current study. E2:T ratios ranged from 0.15 to 1.77 for all male carp used in this study and for males in the same stage of sexual maturation. There were no significant differences in E2:T ratio among male carp held at different sites in Lake Mead (Figure 8). Plasma E2:T ratios ranged from 0.17 to 3.86 for all female carp used in the current study and for female carp in the same stage of sexual maturation. Females held at LW and HF had significantly greater plasma E2:T ratios than females held at both reference sites (Figure 8). E2:T ratios were significantly different between males and females at all sites but reference site WB.
of 15-20 °C, it is possible that the small site-to-site differences in water temperature might have affected sex steroid production.
FIGURE 8. Ratio of plasma 17β-estradiol to plasma testosterone (E2:T) in adult female and male carp. Data are reported only for those carp in the same stage of sexual maturation. Bar heights represent site means, error bars are 2 SEM, and numbers on bars are sample sizes. Parametric statistical analyses were conducted on log transformed and untransformed data for females and males, respectively. Letters above bars represent Tukey groupings within each sex (p < 0.05); i.e., fish at sites with bars marked with common letters were not significantly different with regard to E2:T ratio. LW, Las Vegas Wash; LX, Las Vegas Bay; MC, Moon Cove; WB, Water Barge Cove, HF, Hatchery Fish (hatchery controls). Changes in E2:T ratio can be difficult to interpret in female carp because the ratio can change over the course of gonadal recrudescence. Also, changes in sex steroid profiles and E2:T ratio in relation to the reproductive cycle appear to vary for female carp of different strains and geographic locations, so the general assertion that E:A ratios are greater than 1 for females does not always hold. In a recent assessment of seasonal changes in plasma sex steroid concentrations in female carp, plasma T concentrations were generally greater than plasma E2 concentrations except during fall gonadal recrudescence (62). In the U.K., Manning and Kime found that plasma E2 was greater in female carp with late vitellogenic ovaries, whereas plasma T was greater in the most sexually mature female carp with post-vitellogenic ovaries (42). Likewise, Colombo et al. reported that aromatase activity decreases in the post-vitellogenic ovary in carp (65). E2:T ratios, then, shift from greater than 1 to less than 1 as female carp reach more advanced stages of sexual development. Thus, greater E2:T ratio in female carp held at LW (and perhaps at LX, although the increase is not significant relative to both reference sites) might indicate that they were in a less advanced stage of sexual development, possibly due to adverse effects of LW influent. There were no statistically significant differences in GSI or ovarian follicle development profiles among females at different sites to support that hypothesis. However, because carp have asynchronous ovaries, it is difficult to determine their precise stage of reproductive development, so it is possible that there were slight differences in reproductive timing among fish at different sites that would result in differences in sex steroid profiles that are unrelated to contaminants. Average water temperatures at the exposure sites exhibited a pattern (MC < WB < LX < LW) that paralleled the pattern of median E2:T ratios among female carp held in cages at these sites. Because T is the precursor for synthesis of E2 in the teleost ovary (18), conversion of T to E2 might have increased with water temperature if females were still undergoing vitellogenesis. In female carp in the earlier stages of vitellogenesis, with high circulating concentrations of E2, in vitro E2 production by ovarian fragments increases very significantly from 15 to 20 °C, while for those in the later stages of vitellogenesis, with greater circulating concentrations of T, in vitro T production increases very significantly over the same temperature range (42). Because water temperatures at sites used in this study fall within the range
The post-vitellogenic shift from production of E2 to T was not observed in female carp in a study in China (66) or in a study in Israel (64). In the latter study, plasma E2 and T varied together throughout the reproductive cycle until the post-vitellogenic phase of ovarian development, when plasma E2 concentrations remained at an elevated level during and for several weeks after spawning, while plasma T declined just prior to spawning (64). Likewise, female carp held in aquaria under simulated natural photoperiod had elevated plasma E2 in spring relative to summer and fall (58). If this same pattern of steroidogenesis occurred in female carp caged in Lake Mead and the fish were in a later stage of gonadal recrudescence, the observed steroid concentrations and E2:T ratios might indicate that females at the warmer LW and LX sites were closer to spawning than the females at the reference sites, with plasma E2 concentrations remaining at elevated levels and plasma T beginning to decline. This explanation appears to roughly fit the steroid patterns observed for female carp in this study, with the exception of the generally low steroid concentrations in females at WB. Clearly, a thorough understanding of the pattern of steroidogenesis and its relationship to the reproductive cycle for the specific fish population under study would improve the ability to interpret plasma sex steroid profiles for female carp undergoing gonadal recrudescence. In Vitro T Production by Gonad Tissue. Both male and female carp held in cages at LX had greater basal capacity to produce T in gonad tissue than did fish held at other sites, and ovarian tissue in female carp held at LW had less capacity to produce T than that in females at other sites (Table 3). Since reduced in vitro steroid production is the expected finding in fish with impaired steroidogenic capacity due to exposure to contaminants (18), the reason for increased in vitro T production in fish at LX relative to those at LW, where contaminants are presumably present in greater concentrations, is not clear. However, in vitro T production values for all carp in this study were similar to basal in vitro T production values reported previously for other cyprinids (goldfish and fathead minnows) (18). Likewise, basal in vitro T production for male carp was similar to that reported for adult male carp in a different study (55). The lesser in vitro T production in females at LW did not result in significantly reduced circulating sex steroid concentrations in LW females relative to females at both reference sites, and greater in vitro T production in male and female carp caged at LX did not produce a corresponding increase in plasma T relative to fish at both reference sites. EROD Activity. EROD activity is a measure of the mixed function oxygenase (MFO) activity catalyzed by hydrocarboninducible cytochrome P450 1A proteins (67). EROD activity in fish is commonly used as a biomarker of exposure to certain planar halogenated hydrocarbons, PAHs, and structurally related compounds that bind to the aryl hydrocarbon receptor (68) (i.e., the “dioxin-like” compounds). EROD activity in common carp hepatopancreas is induced by exposure to a variety of dioxin-like compounds and is particularly useful as a biomarker in this species because of the wide range between basal and induced activities (68). Because the MFO system metabolizes both xenobiotics and steroids (69), it has been hypothesized that exposure to chemicals that induce MFO activity might interfere with steroid metabolism in fish (17, 26) such that MFO activity should be considered as a potential modifying factor in field studies where circulating steroid concentrations are end points. However, EROD induction is not consistently related with decreased circulating sex steroid concentrations in fish (68). VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Female and male carp held at LW had significantly greater hepatopancreas EROD activity than those held at all other sites or at both reference sites, respectively (Table 3). In fish, EROD activity and other monooxygenase activities are modulated by many factors other than contaminant exposure, including season, temperature, photoperiod, nutritional status, sex, and reproductive cycles (70, 71), so interpretation of small differences in EROD activity in field studies should be done with caution. The elevation in EROD activity in LW carp is slight since EROD induction less than 10-fold is considered to be weak (68). Basal EROD activity in carp from different studies varies greatly (0-4600 pmol min-1 mg-1) (68), and EROD activities for all carp used in this study fall within that range. EROD activity for male carp held at HF, where they were not exposed to LW influent, was not significantly different from that for males held at LW or LX. Water temperature was greater at LW than at the other sites, and others have noted that EROD activity in carp increased with water temperature (26). Thus, available evidence suggests that dioxin-like contaminants did not have a substantial effect on fish used in this study and that site characteristics such as water temperature might be responsible for observed differences. Biological Significance of Observed Effects. Although plasma VTG was greater in male carp held in cages at LW than in males at the other sites, the difference was small. Exposure to estrogenic contaminants might have caused VTG induction in LW males, but greater water temperature at that site was a potential confounding factor. Furthermore, a causal link between low-level VTG induction and reproductive dysfunction in male fish has not been established, so this cannot be considered a detrimental effect. Plasma E2 was moderately greater in male carp caged at LW and LX than in males at the reference sites, but temperature was a potential confounding factor, and E:A ratios did not indicate an imbalance in the male sex steroid milieu. Other differences among fish caged at different sites are small, potentially explained by temperature differences among sites, and/or not clearly related to concentration of LW influent. Comparison among Studies of Caged Carp and Feral Carp in Lake Mead. Lower plasma E2 and 11-KT were observed in feral male carp captured from LX but not in those captured from LW (3), while increased plasma E2 was observed in male carp caged in LW and LX in the current study. The reason for this difference is not apparent. VTG was detected in the plasma of feral male carp caught from LW and LX but was not detected in males from a reference site (3), and increased VTG also was observed in male carp held in cages in LW in current study. Increased plasma 11KT and decreased plasma E2:11-KT ratio were observed in feral femal carp caught in LW, and increased plasma VTG was observed in feral female carp caught from LX (3), but none of these effects were seen in caged female carp in the current study. No effects of LW influent on GSI or ovarian histology were observed in feral (3, 35) or caged female carp, indicating that any effects were not severe enough to damage ovarian tissue structure or to impair gonadal recrudescence. In the 1995 study of feral male carp (3) and in the current study of caged male carp, no effects on GSI or testicular histology were observed, but another study determined that feral male carp captured from LX had consistently lower GSI and, on one sampling date, a lower proportion of sperm relative to other germ cell stages (35). There are many potential reasons for the inconsistencies between results of the current study and previous studies of feral carp caught from Lake Mead (3, 35). First, the fish used in the current study were exposed only for a limited duration and as sexually differentiated adults, whereas feral fish might have been exposed to contaminants associated with LW during the more sensitive period of sexual differentiation 6394
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and for a longer period of time that allowed for bioaccumulation and delayed effects. Results from the few laboratory studies (50, 60) that sought to characterize the reproductive system responses of adult carp to waterborne EDCs suggest that they are not as sensitive as the adults of some other fish species. Feral carp had access to sediment and natural forage and might have accumulated contaminants through those routes of exposure, which were not available to the caged carp. Contaminants present in LW might have varied qualitatively and quantitively during the different time periods studied. The 1995 study of feral carp (3) was conducted in May, when water temperatures probably were slightly greater than those experienced by carp in the current study and might have allowed for greater production of VTG in response to exposure to estrogenic contaminants. Patin ˜o et al. (35) sampled feral carp repeatedly over the course of two spawning seasons, increasing the likelihood of identifying effects that might be apparent only seasonally. Finally, the caged carp might have lost weight during the exposure period, possibly indicating that the exposure conditions produced some stress that might have affected the responses of the fish. The variation in some end points among carp held in cages at two different reference sites emphasizes the importance of using multiple reference sites whenever it is feasible in field studies where unknown and/or uncontrolled factors might modulate endocrine responses under study.
Acknowledgments The Southern Nevada Water Authority (SNWA) and the U.S. Bureau of Reclamation provided funding, field and laboratory support, and assistance in study design. Peggy Roefer, Kim Zikmund, and Alan Sims of SNWA were particularly helpful. Michigan State University student Peter Horvath assisted with the field work. The National Institute of Environmental Health Sciences provided a Research Service Award (NIH Training Grant ES07255) to E.M.S. The National Park Service (NPS) at Lake Mead National Recreational Area and the Nevada Division of Wildlife (NDOW) provided advice on study design, field support, and facilities for holding fish. Bill Burke (NPS), John Hutchings (NDOW), and the employees of the Lake Mead Fish Hatchery (NDOW) deserve special mention. The NPS volunteers, particularly Fred DeSilva, generously donated their time to operate boats. Dr. Nancy Denslow and Kevin Kroll, University of Florida, provided advice on adaptation of the VTG ELISA.
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Received for review February 27, 2004. Revised manuscript received July 15, 2004. Accepted August 4, 2004. ES049690N
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